Dipeptide PA3264 derived from rare and endangered Squama Manis is a novel bioactive peptide for the treatment of triple-negative breast cancer

Background Squama Manis is a valuable traditional Chinese medicine with a long history of medicinal use in the treatment of breast-related diseases. However, owing to the excessive exploitation and utilization of the resources, Squama Manis has been included in the list of rare and endangered wild animals. The conservation of the resources of Squama Manis and continuing its clinical application has become an urgent problem, and the search for small-molecule substitutes for Squama Manis is an effective way to achieve this goal. Previous studies have identified PA3264 as a possible active ingredient in Squama Manis. In this study, we systematically investigated the pharmacological effects and mechanisms of PA3264 in the treatment of triple-negative breast cancer (TNBC), a representative breast-related disease. Methods Cell viability and colony formation assays were performed after treatment with the target dipeptide PA3264 in vitro. Next, 4T1 orthotopic tumors and humanized PBMC-CDX mouse models were generated to examine the antitumor effect of PA3264 in vivo. Transcriptome sequencing and molecular docking experiments were performed to predict pathways to function. Western blotting and quantitative real-time PCR were used to validate the molecular mechanisms underlying the anticancer effects of PA3264. Results PA3264 significantly inhibited cell viability and migration of breast cancer cells in vitro. Furthermore, PA3264 suppressed the tumor size and reduced the tumor weight in vivo. Finally, it was verified that PA3264 prevented the progression of breast cancer by inhibiting the PI3K/AKT/NF-κB pathway, causing cell cycle arrest, and promoting apoptosis. Conclusions This study elucidated that PA3264 derived from rare and endangered Squama Manis was a novel bioactive peptide for treating triple-negative breast cancer from a scientific research perspective. Supplementary Information The online version contains supplementary material available at 10.1186/s13020-024-00979-x.


Introduction
Squama Manis (pangolin scale) from the scaly armor of Manis pentadactyla Linnaeus has a long history of medicinal use in traditional Chinese medicine.It was first recorded in the "Ming Yi Bie Lu" by Tao Hongjing of the Southern Dynasty and began to be known as "Chuan shanjia" in the classic works of Chinese medicine [1].It has a salty flavor and is mildly cold.It has the function of activating blood and resolving mass, unblocking the meridian and promoting lactation, and dispersing swelling and expelling pus, et al. [2].Squama Manis has been formulated as an ingredient of TCM prescriptions to treat patients with symptoms as amenorrhea, abdominal mass, inhibited lactation, abscess and sore, especially widely used in breast related diseases [3], including mastitis [4], breast hyperplasia [5], and breast cancer [6].
Owing to its significant food and medicinal value, Squama Manis has been subjected to destructive and indiscriminate hunting.Squama Manis is an exceptionally ecologically specialized monotypic order and family, which cannot be successfully reared and bred on a large scale commercially around the globe and can only be captured in the wild, thus leading to a drastic reduction in wild resources [7].Squama Manis is one of the wild medicinal species protected under China's Regulations on the Protection and Management of Wild Medicinal Resources, which has been elevated from the second level of national critical protection to the first level since 2020 and has been classified as critically endangered by the International Union for Conservation of Nature (IUCN) Red List of Threatened Species [7,8].Therefore, it was no longer included in the 2020 edition of the Chinese Pharmacopoeia.Although the clinical efficacy of Squama Manis is exact, it has attracted considerable attention both domestically and abroad because of its endangered resources.Hence, resource protection and the search for alternatives to Squama Manis with similar efficacies are of great significance.
A great deal of work has also been carried out on the chemical composition of Squama Manis and its pharmacological effects, including anticoagulant, antithrombotic, and antitumor [1].It has been reported that cyclic dipeptides are the main active ingredients, and L-serine-L-tyrosyclic dipeptide, D-serine-L-tyrosyclic dipeptide, L-glycine-L-tyrosyclic dipeptide, and other constituents have been successfully isolated from Squama Manis [9].The discovery of more active peptides for treating breastrelated diseases and the exploration of their mechanisms need to be followed by further studies.Previous studies have identified a peptide, PA3264, as a possible active ingredient of Squama Manis, but its pharmacological activity in breast diseases is unknown.
Triple-negative breast cancer (TNBC), a typical breast disease, is an aggressive subtype of breast cancer with recurrence and metastasis and has a poor prognosis [10].The PI3K/AKT pathway has been reported to be a frequent signaling pathway in TNBC, making it one of the most important signaling pathways for therapeutic intervention.In addition, abnormal activation of nuclear factor-κappa B (NF-κB) induces the expression of target genes that participate in cell cycle regulation, cell proliferation, and apoptosis [11,12].Thus, inhibition of the PI3K/AKT pathway, as well as the downstream NF-κB pathway, contributes to antitumor effects.Here, a new linear dipeptide, PA3264, from Squama Manis, had a significant inhibitory effect on TNBC.Mechanistically, it inhibited the proliferation of breast cancer cells, caused cell cycle arrest, and promoted apoptosis by inhibiting the PI3K/AKT/NF-κB pathway.This study provides new ideas for the search for Squama Manis substitutes and the conservation of its resources.It provides a theoretical basis for a novel and effective leading compound, PA3264, for treating TNBC.

Materials
D-Tyrosine-L-serine (PA3264) was synthesized and purified by GenScript Company (C216JHB100_5, NJ, USA).The HPLC purity was ≥ 98.0%.The peptide was soluble in ddH 2 O and stored at −20 °C.A patent application was filed for the compound (Chinese patent application for authorization No.: CN 114315958 B).

UPLC-MS/MS analysis of PA3264 from Squama Manis
Squama Manis was obtained from Fifth Medical Center of Chinese PLA General Hospital and identified by Xiaohe Xiao professor.A Vanquish Flex UPLC system (Thermo Scientific, MA, USA) interfaced with a Q Exactive Focus mass spectrometer (Thermo Scientific) was used for UPLC-MS/MS analysis [13][14][15].The following UPLC condition was used: The chromatographic column a Thermo Scientific Accucore HILIC column (2.1 × 100 mm, 2.6 μm, Part No.: 17526-102130); and an eluent of 10 mM ammonium acetate in 10% acetonitrile (A) and 10 mM ammonium acetate in acetonitrile (B) with the following gradient elution system: 25% A and 75% B in 0-6 min, then 45% B in 6-7 min, 30% B in 7-10.1 min, 75% B in 10.1-22 min at an eluent flow rate of 0.3 mL/min.The column temperature was maintained at 40 °C.The total running time was 22 min.An injection volume of 5 μL was used for each sample.The optimized parameters of MS were as follows: Spray voltage, 3.8 kV; Aux gas heater temperature, 350 °C; Sheath gas flow rate, 45 arb; Aux gas flow rate, 15 arb; Capillary temperature, 320 °C; S-lens RF, 60 V; scan mode: (1)

Cell cultures
The breast cancer cell lines MDA-MB-231 and 4T1 were obtained from ATCC and maintained in high-glucose Dulbecco Modified Eagle Medium (DMEM) and RPMI 1640 medium supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin (all from BOSTER, Beijing, China), and 10% fetal bovine serum (C04001-500, VivaCell, Beijing, China) in a humidified atmosphere of 5% CO 2 at 37 °C for serial passaging.The cells were harvested during the logarithmic growth phase for subsequent experiments.The cells were frozen in a serum-free cell freezing medium (03.17004DA,EallBio, Beijing Eall-Bio Biomedical Technology Co., Ltd, China).

Mice
Female BALB/c mice (6-8 weeks old; 18-20 g) were purchased from SPF Biotechnology Co., Ltd.(Beijing, China) and housed in a standard pathogen-free (SPF) standard room.Six-week-old female supra-immunodeficient NCG-hIL15 mice were purchased from GemPharmatech (Jiangsu, China) and were maintained in microisolator cages under pathogen-free conditions.Laboratory animal production license: SCXY (Jing) 2019-0010, Laboratory animal production license: SCXK (Su) 2020-0004.All mice were given free access to standard laboratory chow and water ad libitum for the experiment with a 12-h light-dark cycle at a temperature of 21-25 °C.Protocols for animal experiments were approved by the guidelines for the care and use of laboratory animals.All experiments were performed under the approved guidelines of the Animal Ethics Committee of the Fifth Medical Center, Chinese PLA General Hospital (IACUC-2023-0014).

Tumor challenge and treatment experiments
4T1 orthotopic tumors were induced via subdermal inoculation of 2 × 10 5 4T1 early passage cells suspended in 100 μL of PBS into the 4th mammary fat pad of BALB/c females, as described previously [16].At 1-week postinoculation (tumors were palpable around 15 mm 3 ), the animals were randomly distributed into the control and treatment groups.PA3264 (100 mg/kg) was administered intraperitoneally (i.p.) once daily.Cisplatin (HY-17394, MedChemExpress, NJ, USA) was used as the positive control (2 mg/kg, once every 4 days) [17].Animal body weight and tumor growth were measured three times per week.Tumor volume was calculated using the following equation: 0.5 × length × width 2 [18].At the end of the experimental period, the mice were sacrificed, and the remaining tumors were harvested for weighing and other experiments.
The humanized PBMC-CDX mouse model was established previously [19,20].MDA-MB-231 tumor cells were subcutaneously inoculated into tumor-donor mice.After the tumors grew, tumors with a volume of approximately 500-1000 mm 3 were removed under aseptic conditions, cut into tumor masses of approximately 8 mm 3 , and inoculated subcutaneously in the right flank of experimental mice with a cannula needle.Each mouse was injected with a tumor mass.Three days after tumor inoculation, healthy adult PBMCs were inoculated into the mice by resuspension in PBS at an inoculation rate of 2 × 10 6 /mouse.Tumors were administered in groups when they grew to approximately 50-70 mm 3 (Day10).PA3264 was intraperitoneally injected at a dose of 50 mg/ kg for 21 days.

Transcriptome sequencing (RNA-seq) analysis
Total RNA was extracted from the samples and enriched with oligo (dT)-attached magnetic beads.The synthesized and purified double-stranded cDNA was subjected to end repair and 3'-adenylation.Sequencing adapters were connected to the ends of these 3′ adenylated cDNA fragments, and the selected fragments were amplified by PCR and qualified using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).Libraries were constructed using the VAHTS Universal V6 RNAseq Library Prep Kit according to the manufacturer's instructions after the quality inspection was completed.Sequencing was performed using Illumina HiSeqTM 2500.Differential expression analysis was performed using the DESeq2.A Q value < 0.05 and foldchange > 2 or foldchange < 0.5 was set as the threshold for significantly differentially expressed genes (DEGs).Transcriptome sequencing and analysis were performed by OE Biotech Co. Ltd. (Shanghai, China) using standard procedures.

Molecular docking
Molecular docking was performed using Schrödinger Suite Release 2018-1 [23,24].PI3Kγ, AKT1, and NF-κB p65 proteins were used as receptors, and PA3264 was used as a ligand.Three-dimensional crystal structures of PI3Kγ (PDB ID: 4HVB), AKT1 (PDB ID: 4EKL), and NF-κB p65 (PDB ID: 1NFI) were retrieved from the RCBS PDB database.This structure was optimized before docking using Protein Preparation Wizard in Maestro 11.5.The docking of the receptor (target) preparation involves the deletion of water molecules from target proteins, removal of protein polymorphisms, complementation of non-complete amino acid residues, and hydrogenation of proteins, followed by lattice-box construction of each target protein.LigPrep was used to convert structures from 2-dimensional to 3-dimensional, correction improper bond distances and bond orders, generating ionization states, and minimizing energy.The processed protein and ligand structures were subjected to docking analysis.Finally, docking scores were calculated to evaluate the binding activity between the ligand and three targets.The display diagram of the molecular docking results was modified using PyMOL.

Cell viability assay
To assess the effects of PA3264 on cell viability, 1.5 × 10 5 MDA-MB-231 and 9.0 × 10 4 4T1 cells per well in 96 well plates were incubated for 12 h.The medium was then replaced with different PA3264 concentrations (0, 0.078, 0.156, 0.313, 0.625, 1.25, 2.5, 5, 10, and 20 mg/mL) for the indicated times.Cell viability was measured using the CCK-8 assay kit (G4103-5ML, Servicebio, Wuhan, China) according to the manufacturer's instructions.The optical density (OD) values used as the index of cell viability were measured at 450 nm using a microplate reader, and the IC 50 values were calculated as described previously [21].Lactate dehydrogenase (LDH) is a stable cytoplasmic enzyme in all cells.LDH is rapidly released into the cell culture supernatant when the plasma membrane is damaged, a key feature of cells undergoing apoptosis, necrosis, and other forms of cellular damage [25].The LDH release assay was performed using the LDH Cytotoxicity Assay kit (G1610-100 T, Servicebio, Wuhan, China), according to the manufacturer's protocols.

Colony formation assay
To assess clonogenic ability, single cell suspensions containing 2 × 10 3 MDA-MB-231 and 4T1 cells were seeded in 12-well plates and allowed to adhere for 24 h, and treated with PA3264 for 5 days.The cells were fixed in 4% paraformaldehyde and stained with crystal violet.Finally, the colonies were quantified using the ImageJ software.

Wound healing assay
A total of 2 × 10 5 cells were seeded in the wells of a 12-well plate for 24 h.The cell layer was scratched using a sterile pipette tip (200 µL).Subsequently, cells were cultured with the indicated concentrations of PA3264.Representative images of the wounds were taken at 0 and 24 h using an Eclipse microscope (Nikon Corporation, Tokyo, Japan).Each experiment was conducted in triplicate.

Quantitative real-time PCR (qRT-PCR)
Total RNA was extracted using TRIzol reagent (Alcatel, Beijing, China), and the isolated RNA was reversetranscribed into cDNA using StarScript III All-in-one RT Mix with gDNA Remover (GenStar, Beijing, China).Primer sequences used are listed in Table 1.Quantitative real-time PCR was performed on a QuantStudio 6 (96-well format, Thermo Fisher, USA) using SYBR Green qPCR Master Mix (Low ROX) (GenStar, Beijing, China).The PCR conditions were as follows: 95 °C for 5 min, followed by 40 cycles of 95 °C for 10 s, and 60 °C for 60 s.The expression levels of these genes were normalized to that of β-actin.The data were analyzed and displayed using the ΔΔCt or 2 −ΔΔCt method.

Quantification and statistical analysis
Two-tailed unpaired Student's t-test was used to compare two groups.Multiple groups were tested using a one-way analysis of variance (ANOVA).Two-factor ANOVA was used for tumor growth analysis.All data are presented as mean ± SEM.Data were processed using GraphPad Prism version 9 (GraphPad Software).In all cases, p values of 0.05 and below were considered to be statistically significant: p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***) were considered statistically significant.Detailed information regarding the number of replicates can be found in the figure legend.

UPLC-MS/MS analysis of PA3264 from Squama Manis
The extracted ion chromatogram of PA3264 from Squama Manis showed that its molecular formula of PA3264 was C12H16N2O5, with a theoretically calculated [M + H] + mass charge ratio (m/z) of 269.1132.The lower image characterized the possible structures of the two fragment ions in the mass chromatogram of PA3264 (Fig. 1A).The mass spectral verification of the synthetic dipeptide PA3264 was shown in Fig. 1B.The theoretically calculated [M + H] + mass-to-charge ratio (m/z) was 269.11289.One of the fragment ions matched the one shown in Fig. 1A.The structure of PA3264 was roughly deduced from the molecular ion peaks and fragment ion results of PA3264 in Squama Manis and synthetic peptide samples.Next, we investigated the pharmacological activity and mechanism of action of PA3264 in breastrelated diseases represented by triple-negative breast cancer.

PA3264 restrained the viability and clonogenicity in murine and human-derived breast cancer cells
To explore the effect of PA3264 on the proliferation of murine and human breast cancer cells, CCK-8 and LDH release assays were used to determine the viability of 4T1 and MDA-MB-231 cells after treatment with various concentrations of PA3264 for 24 h.The results showed that the proliferation of 4T1 and MDA-MB-231 cells treated with PA3264 was significantly lower than that of the control group, in a dose-dependent manner (Figs.2A  and 3A).The LDH release assay showed that PA3264 treatment accelerated plasma membrane damage in breast cancer cells in a dose-dependent manner (Figs.2B  and 3B).In addition, colony formation assays showed a decrease in the number of colonies formed as the intervention concentration of PA3264 increased (Figs. 2C, D  and 3C, D).These results indicated that PA3264 curbed the cell viability and colony formation of murine and human-derived breast cancer cells in a dose-dependent manner.

PA3264 constrained the migration of 4T1 cells
To evaluate the effect of PA3264 on 4T1 cell migration, wound healing assays were performed.After 4T1 cells were incubated with 20 mM PA3264 for 24 h, the migration distance was significantly reduced by 39% compared with the control group (Fig. 2E, F, p < 0.05).The data demonstrated that PA3264 had a remarkable prevention on wound healing as an inhibitor of 4T1 cell migration.

PA3264 inhibited tumor growth in vivo
To examine the antitumor effects of PA3264 in vivo, 4T1 orthotopic tumors were constructed and treated.As shown in Fig. 4A, B, tumor volume and weight in the model group increased more dramatically.At the same time, cisplatin and PA3264 treatments significantly inhibited the increase in tumor volume and weight as early as 4 weeks after injection.At the end of this period, the tumor volume and weight in PA3264-treated mice were reduced by 41% and 32%, respectively, compared to those in control mice (p < 0.01).Cisplatin used as a positive control had a significant depression effect on tumor size and weight.Strikingly, the treated groups had a smaller burden than the control group (Fig. 4E).In addition, a humanized PBMC-CDX mouse model was used to validate the antitumor activity of the PA3264.Tumor volume was also reduced in mice treated with PA3264 for 24 consecutive days compared with the control group (Fig. 4D, p < 0.01).
Body weight is one of the most important indicators for evaluating the safety of anticancer drugs.Throughout the experimental period, the body weight of mice treated with PA3264 did not change significantly compared with that of the control group (Fig. 4C).However, the body weights of cisplatin-treated mice decreased significantly 3 weeks after injection compared with the control group (Fig. 4C).Additionally, the effect of PA3264 on liver and renal function indicators in 4T1 tumor-bearing mice was investigated by measuring serum related biochemical parameters (ALT, AST, BUN, and CRE).Compared with the normal group, the serum BUN levels in the model group were significantly elevated (p < 0.01), indicating that the renal function of the tumor-bearing mice was damaged to a certain extent (Suppl.Figure 1C).After cisplatin treatment in the tumor-bearing mice, the levels of serum ALT, BUN, and CRE in the cisplatin group were significantly higher than those in the control group (p < 0.05, Suppl.Figure 1A, 1C, and 1D), while the levels of AST were also increased (p > 0.05, Suppl.Figure 1B), indicating that the liver and renal function of the tumor-bearing mice were damaged to a certain extent by cisplatin administration.After treatment with PA3264 in the tumor-loaded mice, the levels of ALT, AST, BUN, and CRE were lower than those in the model group, but the difference was not statistically significant (p > 0.05, Suppl.Figure 1A-D).Compared with the cisplatin group, the levels of ALT, BUN, and CRE in the PA3264-treated  1A, 1C, and 1D), while the levels of AST were also reduced.Still, the difference was not statistically significant (p > 0.05, Suppl.Figure 1B).This suggested that PA3264 did not significantly damage the liver and kidney function of the tumor-loaded mice.In summary, treatment with PA3264 and cisplatin was largely effective in regressing tumors and PA3264 was safer to administer in vivo than cisplatin.
The histology of tumor sections showed that tumor cells, which had apparent nucleoli and large nuclei, were arranged tightly in the control group.In contrast, there was a loose arrangement in the PA3264 treated group (Fig. 4F).In addition, immunohistochemical staining of tumor tissues with Ki67 and Caspase 3 was performed.
PA3264 administration decreased the expression of Ki67 and increased the expression of Caspase 3 in the tissues compared with the control group (Fig. 4G).Quantitative real-time PCR analysis of tumor tissues showed that the expression of Bax and the Bax/Bcl-2 ratio was upregulated after PA3264 treatment (Fig. 4H).Taken together, these in vivo data suggest that PA3264 inhibited the proliferation and promoted the apoptosis of triple-negative breast cancer.

Identification of DEGs and their pathways in PA3264-mediated tumor suppression by RNA-seq analysis
To explore the genes and pathways involved in inhibiting 4T1-induced triple-negative breast cancer by PA3264, RNA from the four replicate samples from the control and PA3264-treated groups were sequenced.Cutoff values were set at |log2 fold change (FC)|> 1 and p < 0.05.A clustering heatmap and volcanic distribution of the DEGs were obtained.A total of 226 differential expression genes (DEGs) were identified.Overall, 90 upregulated and 136 downregulated DEGs were identified in the control and PA3264-treated groups, respectively (Fig. 5A).Hierarchical clustering analysis of the DEGs between the control and PA3264-treated groups was presented as a heat map, indicating significant differences in the expression patterns of the DEGs between the two groups (Fig. 5B).The DEGs were subjected to GO enrichment analysis to characterize their functions.Results showed that these DEGs were significantly enriched in GO functions associated with extracellular regions, extracellular space, neutrophil chemotaxis, and cysteine-type endopeptidase inhibitor activity (Fig. 5C).Moreover, KEGG pathway analysis showed that the DEGs were mainly associated with cytokine-cytokine receptor interaction, NF-kappa B signaling pathway, ECM-receptor interaction, PI3K-AKT signaling pathway, IL-17 signaling pathway, and chemokine signaling pathway in control vs. PA3264-treated groups (Fig. 5D).Furthermore, some DEGs related to the PI3K/AKT signaling pathway with significant fold differences in expression were verified by qRT-PCR.The results were consistent with the RNA-seq results (Fig. 5E).

Molecular docking
Aberrant activation of the PI3K/AKT/NF-κB pathway is commonly observed in breast cancer, resulting in the increased proliferation and invasive potential of cancer cells.To predict the binding affinity of PA3264 for related proteins in the PI3K/AKT/NF-κB pathway, molecular docking studies were performed.PA3264 and the surrounding amino acid residues of macromolecular targets helped stabilize the complex structure, thus enhancing binding affinity.Figure 6A, B depict the interaction of PA3264 with the binding site residues of PI3Kγ.The phenyl ring of Tyr867 interacted with PA3264 via face-to-face pi-pi stacking interaction with the phenyl ring of PA3264.Additionally, the hydroxy group of PA3264 formed 3H-bonds with residues Asp967, Lys833, and Val882.Next, the molecular docking of PA3264 with AKT1 (Fig. 6C, D) and NF-κB p65 (Fig. 6E, F) was carried out.Furthermore, the molecular docking results revealed that PA3264 had the highest binding affinity with the inhibitory targets of PI3K, with a docking score of −6.987, as shown in Table 2.

PA3264 caused cell cycle arrest and promoted apoptosis by regulating the PI3K/AKT/NF-κB signaling axis
To investigate the anticancer effects of PA3264 and verify the PI3K/AKT/NF-κB pathway-dependent mechanism, we measured the expression levels of related proteins using western blotting.As shown in Fig. 7A-F, following intervention with different concentrations of PA3264 for 24 h on the 4T1 cells, the protein expression of p-PI3K, p-AKT, p-p65, and Cyclin D1 was reduced in a dosedependent manner.And the expression levels of p-PI3K, p-AKT, p-p65, and Cyclin D1 proteins were significantly lower in the 20 mM and 40 mM groups than in the control group (p < 0.01 or 0.05).Consistent with the WB analysis, the mRNA expression levels of Bcl-2 and Cyclin D1 also gradually declined in PA3264-treated 4T1 cells compared to those in the control group (p < 0.01 or 0.05, Fig. 7G, H), as expected.To further validate these results, we performed the same experiments using MDA-MB-231 cells.The expression levels of p-PI3K, p-AKT, p-p65, and Cyclin D1 proteins were significantly lower in the 10 mM and 20 mM groups than in the control group (p < 0.01 or 0.05, Suppl.Figure 2A-F).Moreover, PA3264 treatment significantly downregulated Bcl-2 and Cyclin D1 expression in the 10 mM and 20 mM groups compared to that in the control group (p < 0.01 or 0.05), as evidenced by the mRNA expression level (Suppl.Figure 2G-H).Overall,  To further validate the mechanism of PA3264 in the treatment of TNBC, PI3K and NF-κB pathway inhibitors were used in western blotting experiments.LY294002 and JSH-23 have been reported to be PI3K and NF-κB pathway inhibitors, respectively [26][27][28][29].We investigated the inhibitory effects of PA3264 in combination with LY294002 on PI3K/AKT signaling and JSH-23 on NF-κB signaling in breast cancer cells (MDA-MB-231 and 4T1).LY294002 and PA3264 reduced p-PI3K, p-AKT, and p-p65 protein expression in comparison with the control group (Fig. 7I-M and Suppl.Figure 2I-M).Compared with the LY294002 group, the expression of p-PI3K, p-AKT and p-p65 proteins decreased with no significant difference, when PA3264 was co-administered with LY294002.Still, Cyclin D1 protein expression had a significant decreasing trend (p < 0.01, Fig. 7N and Suppl.Figure 2N), which indicated that PA3264 exerted a part of its anti-TNBC effect by inhibiting the PI3K/AKT signaling pathway.In addition, JSH-23 and PA3264 inhibited the expression of p-p65 and Cyclin D1 proteins in comparison with the control group.The combination of PA3264 and JSH-23 reduced the expression of p-p65 and Cyclin D1 proteins without significant difference compared with the JSH-23 group, suggesting that PA3264 played a part of its anti-TNBC effect by suppressing the NF-κB signaling pathway.Therefore, in this study, we found that PA3264 exerted a certain anti-TNBC effect by inhibiting the PI3K/AKT/NF-κB signaling pathway.

Discussion
Squama Manis has a long history of medicinal application in China to invigorate blood circulation and remove blood stasis.Clinically, Squama Manis is used to treat a wide range of diseases with significant results, not only for breast-related diseases but also for other gynecological disorders, including fibroids [31] and infertility [32].Although the effect of Squama Manis has been demonstrated through the accumulation of real-life experiences, the role of individual biochemically active ingredients has not been extensively investigated.Further research should be conducted to develop active compounds as alternatives to Squama Manis to protect this endangered species.
Breast cancer is one of the most common breast-related diseases with a high incidence and mortality rate.TNBC, the most aggressive subtype, quickly metastasizes, and exhibits strong drug resistance.Chemotherapeutic agents are widely used to treat patients with advanced BC.However, approximately 50% of the patients rapidly develop acquired resistance [30].Therefore, it is still important to discover new TNBC treatments, and providing more effective treatments with fewer side effects is the primary and challenging research direction.Traditional Chinese medicine (TCM) plays an anticancer role by regulating the immune system, anti-tumor angiogenesis, tumor cell apoptosis, autophagy dysfunction, and other mechanisms.An increasing body of literature reports traditional Chinese medicine and its active constituents possess significant anticancer activity, such as Icariside II [31] and curcumin [32,33].Therefore, exploring new anticancer agents from TCM and their mechanisms in treating TNBC has become a research hotspot in recent years.
It is widely believed that restraining the PI3K/AKT/ NF-κB pathway is a crucial target for treatment.The omnipresent PI3K/AKT pathway is involved in apoptosis, proliferation, and differentiation by regulating a wide range of target proteins, including NF-κB [34].The phosphorylation of p65 leads to NF-κB translocation into the nucleus and binding to specific sequences in the promoter regions of target genes encoding mediators such as Cyclin D1, Bcl-2 [35,36].By promoting Cyclin D1 and Bcl-2 expression, the NF-κB pathway drives cell cycle progression and promotes the proliferation of cancer cells.Previous studies have found a peptide, PA3264, derived from Squama Manis, but its pharmacological activity in breast diseases is unknown.In this study, we found that PA3264 significantly inhibited the viability of BC cells (MDA-MB-231 and 4T1) in a dose-dependent manner.In addition, mechanistic studies have suggested that PA3264 downregulated the phosphorylation of PI3K, AKT and p65, and decreased the expression of Cyclin D1 and Bcl-2, thereby inhibiting cell proliferation and inducing apoptosis.Consistent with the results of the in vitro experiment, PA3264 significantly inhibited tumor development and induced apoptosis in vivo.Treatment with PA3264 did not substantially affect the average body weight, the liver and kidney function of mice.Taken together, these results suggest that PA3264 is a potentially safe and effective anticancer treatment for TNBC.
There are two main limitations with regard to this research.The present study yielded that PA3264 inhibited cell migration only at 4T1.However, the in vivo effects need to be further investigated.Transcriptome sequencing results enriched multiple signaling pathways that may inhibit TNBC, but we focused only on the PI3K/AKT and NF-κB pathways.The current results suggest that PA3264 inhibited the activation of the PI3K/AKT/NF-κB pathway by suppressing PI3K phosphorylation.To further support this, PI3K and NF-κB pathway inhibitors were used in western blotting experiments.Therefore, in this study, we found that PA3264 exerted a certain anti-TNBC effect by inhibiting the PI3K/AKT/NF-κB signaling pathway, but other mechanisms of action also exist.More studies are needed to address these limitations in the future to characterize its therapeutic functions and elucidate other molecular mechanisms that exert its biological effects in TNBC.

Conclusion
In this study, we preliminarily clarified the anti-tumorigenic activity and mechanisms of action of PA3264 in TNBC treatment.Our findings suggested that PA3264 inhibited tumor growth by modulating the PI3K/AKT/NF-κB signaling axis, suppressing proliferation and promoting apoptosis.Altogether, this study indicates that PA3264 as a potential leading compound has a therapeutic benefit against TNBC and promises to be a substitute for rare and endangered Squama Manis in the treatment of TNBC.

Fig. 1 Fig. 2
Fig. 1 UPLC-MS/MS analysis of PA3264 from Squama Manis.A The upper panel shows the extracted ion chromatogram of PA3264.The mass spectra of PA3264 are shown in the lower panel.B MS and MS/MS spectra of synthetic dipeptide PA3264

Fig. 3
Fig. 3 PA3264 significantly suppressed the viability of MDA-MB-231 cells.A MDA-MB-231 cells were treated with serial concentrations of PA3264, and the effects of PA3264 on cell proliferation were measured using the Cell Counting Kit-8 examinations at 24 h.B LDH was released from MDA-MB-231 cells after 24 h of treatment with different concentrations of PA3264.C-D Images and results of colony formation assay with MDA-MB-231 cells treated with PA3264.Data were presented as mean ± SEM, n = 3, *p < 0.05, **p < 0.01, ***p < 0.001 vs. the control group

Fig. 6
Fig. 6 PA3264 docked in the binding sites of macromolecular targets.i) PA3264 docked to the binding site of PI3K (PDB ID: 4HVB).A 3D picture depicting the binding mode.B 2D interaction diagram showing ligand-receptor interactions; ii) PA3264 docked at the binding site of AKT (PDB ID: 4EKL) C 3D binding pose, D 2D interaction diagram; iii) PA3264 docked at the binding site of NF-κB p65 (PDB ID: 1NFI) E 3D binding pose, F 2D interaction diagram

Fig. 7 Fig. 7 (
Fig.7PA3264 caused cell cycle arrest and induced apoptosis in 4T1 cells via the PI3K/AKT/NF-κB signaling pathway.A-B Dose-response effects of PA3264 on signaling pathways in 4T1 cells treated with the indicated concentrations for 24 h.The levels of p-PI3K, PI3K, p-AKT, AKT, p65, p-p65, and Cyclin D1 in PA3264-treated 4T1 cells were examined using western blotting.C-E The relative intensities of all phosphorylated proteins were calculated after normalization to the total proteins.F Relative intensities of Cyclin D1 were calculated after normalization to HSP90 expression.G-H The expression of Cyclin D1 and Bcl-2 mRNA in 4T1 cells was verified by qRT-PCR.I-J 4T1 cells were treated with 20 mM PA3264, 0.1 μM LY294002, and 10 μM JSH-23 for 24 h.The expression levels of PI3K, AKT1, p65, their phosphorylated forms, and Cyclin D1 were assessed in cell lysates by western blotting analysis.K-M Relative intensities of all phosphorylated proteins were calculated after normalization to total protein.N The relative intensity of Cyclin D1 was calculated after normalization with HSP90 expression.Data were presented as the mean ± SEM (n = 3).*p < 0.05, **p < 0.01, and ***p < 0.001, compared with levels in the untreated group or the group administered alone (See figure on next page.)

Table 1
Primer sequences of target genes these results indicated that PA3264 suppressed the PI3K/ AKT/NF-κB signaling pathway to regulate the cell cycle and apoptosis in breast cancer cells, further validating the accuracy of RNA-seq analysis and molecular docking results.

Table 2
Docking analysis results for the compounds